Oral Anti-Protozoiasis Vaccines Based on Transgenic Plants

ABSTRACT

Leaves of potato plants transformed with a vector that expresses an R7 immunogenic protein gene derived from second-generation schizonts of  Leucocytozoon caulleryi were administered to chickens as an oral vaccine. As a result, the present inventors succeeded in increasing the antibody titer of the chickens.

TECHNICAL FIELD

The present invention relates to plant-expressed oral anti-protozoiasisvaccines that use protective antigens against protozoiasis development.

BACKGROUND ART

Chicken leucocytozoonosis is a disease resulting from infection byLeucocytozoon caulleryi belonging to the suborder Haemosporina. L.caulleryi was recognized for the first time in 1909 in Vietnam in theblood of chicken as round gametocytes that do not carry malariaparticles, and was named as a protozoan belonging to the genusLeucocytozoon.

In Japan, chicken leucocytozoonosis was confirmed for the first time in1954 in Hyogo prefecture. The pathogen was identified as L. caulleryi,and thereafter, experiments demonstrated that this protozoan isbiologically transmitted by a type of blood-sucking insect, Culicoidesarakawae (hereinafter abbreviated as C. arakawae) belonging to the orderDiptera and family Ceratopogonidae. The life cycle of this pathogen hasbeen almost fully elucidated. Leucocytozoon caulleryi has three stagesof development: schizogony (multiplicative reproduction) and gametogony(gamete formation) in the body of the chicken host, and sporogony (sporeformation) in the body of the C. arakawae transmitter.

When C. arakawae infected with this protozoan sucks blood, sporozoitesthat enter the body of chicken with the saliva become parasitic onvascular endothelial cells, mainly in the lung, liver, spleen, kidneyand such, and form first-generation schizonts. On the fifth to seventhday after infection, first-generation merozoites are released into theblood. These first-generation merozites reenter the vascular endothelialcells distributed throughout the body, grow into second-generationschizonts, leave the host cells and grow in the intercellular space inthe late stage. On the 14th day, second-generation merozoites arereleased into the blood by these schizonts. The merozoites entererythroid cells and grow, and on the 18th to 19th day, they separatefrom the host cells and mature as macrogametocytes (female gametocytes)and microgametocytes (male gametocytes).

When C. arakawae sucks blood, both gametocytes are taken up into themidgut together with the blood of the chicken. These gametocytes formmacrogametes (female gametes) and microgametes (male gametes), whichthen conjugate (fertilization) to form zygotes that subsequently formookinetes which enter the intercellular space in the midgut wall anddevelop into oocysts after migrating to beneath the outer membrane ofthe midgut wall. Several tens of sporozoites are formed inside theoocysts, and as the wall of the oocysts breaks down, sporozoites arereleased into the celom of C. arakawae. They then migrate to thesalivary gland to complete the developmental cycle.

Symptoms and lesions in chicken caused by this protozoan infection areobserved mainly in the late stages of schizogony when second-generationschizonts grow, and in the gametogony stage when second-generationmerozoites become parasitic on erythroid cells and develop intogametocytes. Anemic symptoms and bleeding lesions in various organs andtissues, which are most characteristic of this disease, are thought tobe triggered by vascular emboli-accompanying bleeding caused bysecond-generation schizonts, and disruption of red blood cells due toparasitization by protozoans in the gametogonic stage.

Since C. arakawae, the mediator of the protozoan, inhabits wetlands,this infection has been observed to be prevalent in Southeast Asia,China, North Korea, and South Korea where paddy fields are found. InJapan, since the first observation of leucocytozoonosis, the epidemichas recurred every summer and inflicted a significant loss on poultryproductivity. However, the occurrence sharply decreased after feedsstarted to be supplemented with pyrimethamine (since 1964) and a mixtureof pyrimethamine and sulfa drugs (since 1968) as preventive agents.Since then however, acquired resistance of protozoans and residualityissues have restricted the duration of use of effective drugs, and theuse of some of these drugs have been banned for safety reasons. Suchrestrictions are making the prevention of the disease difficult and thedamages are also becoming constant issues.

Conventional experimentally produced vaccines against chickenleucocytozoonosis include live vaccines that use sporozoites,inactivated vaccines in which the antigen is a parasite-derivedsubstance, and inactivated vaccines produced out of organ emulsions ofprotozoan-infected chicken. In the case of live vaccines usingsporozoites, chickens are immunized with a small number of sporozoitesseparated from the salivary glands of C. arakawae [see Non-PatentDocument 1]. Examples of inactivated vaccines in which the antigen is aparasite-derived substance are as follows.

Examples of parasite-derived antigens include first-generationschizonts, first-generation merozoites, second-generation schizonts,second-generation merozoites, and soluble antigens derived from the bodyof chicken and from developing chick embryos. Of these, soluble antigensdetected in the serum of infected chicken on the tenth to fifteenth dayafter sporozoite inoculation have been observed to have nearly the sameantigenic characteristics as second-generation schizonts, and arelatively strong immunogenicity similar to that of thesecond-generation schizonts. Utility has been confirmed for experimentalvaccines produced by inactivating such serum-derived antigenic materialwith formalin [see Non-Patent Document 2].

Furthermore, inactivated vaccines made from organ emulsions weredeveloped to improve the difficulties of obtaining large amounts of theabove-described protozoan antigens. The vaccines were experimentallyproduced out of organ emulsions collected from the thymus, lung, liver,spleen, kidney, and F sac from infected chicken on the thirteenth dayafter sporozoite inoculation, and then inactivated with formalin. Theeffects of such vaccines have been confirmed [see Non-Patent Document3].

To enable a stable and abundant supply of vaccines, the presentinventors have provided vaccines that use genetically modifiedmicroorganisms made to express immunogenic proteins derived fromsecond-generation schizonts of Leucocytozoon caulleryi [see PatentDocument 1].

However, with vaccines developed so far, there is the premise that theywill be administered by injection, and is problematic in that their useis limited due to cost and inconvenience.

-   [Patent Document 1] Granted/Registered Japanese Patent No.3582663-   [Non-Patent Document 1] Shiihara et al., “Shizuokaken Yokei Shikenjo    Kenkyu Hokoku (Bulletin of Shizuoka Poultry Experiment Station)”    13:25-27 (1978)-   [Non-Patent Document 2] Isobe and Suzuki, Jpn, J. Parasitol.,    37:214-219 (1988)-   [Non-Patent Document 3] Morii, T. et al., J. Parasitol. Res.,    76:630-632 (1990)

DISCLOSURE OF THE INVENTION Problems To Be Solved By the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide anti-protozoiasisvaccines that are economical and can be conveniently administered. Morespecifically, an objective of the present invention is to provide oralanti-protozoiasis vaccines with plant-expressed protective antigensagainst protozoiasis development.

Means To Solve the Problems

To solve the above-mentioned issues, the present inventors developedoral anti-protozoiasis vaccines utilizing transformed plants.Specifically, the R7 immunogenic protein gene derived fromsecond-generation schizonts of Leucocytozoon caulleryi (Japanese PatentApplication Kokai Publication No. (JP-A) H07-284392, andGranted/Registered Japanese Patent No. 3582663), was selected as antigengene, and a vector expressing this gene was introduced into potatotubers via Agrobacterium to produce regenerated potato plants from thetubers. Next, the leaves of the transformants were dried and mixed withformula feed for poultry, which was orally administered to chickens thathad been vaccinated beforehand with a R7 immunogenic protein derivedfrom second-generation schizonts of Leucocytozoon caulleryi obtained byexpression in E. coli. As a result, the oral administration successfullyincreased antibody titer in chickens. Increase in antibody titer wasparticularly significant in chickens having a high antibody titer levelat the start of the experiment. Similar techniques can be applied toother protective antigens against protozoans.

That is, the present invention relates to oral anti-protozoiasisvaccines that use plant-expressed protective antigens againstprotozoiasis development, as well as production and uses thereof. Morespecifically, the present invention provides:

-   [1] a vector comprising a gene encoding a protective antigen against    protozoiasis development, wherein the gene is operably linked    downstream of a promoter that can be transcribed in a plant cell;-   [2] a transformed plant cell introduced with a gene encoding a    protective antigen against protozoiasis development;-   [3] a transformed plant comprising the transformed plant cell of    [2];-   [4] a transformed plant which is a progeny or clone of the    transformed plant of [3];-   [5] a propagation material of the transformed plant of [3] or [4];-   [6] a processed material or an extract of the transformant of [3] or    [4], or a propagation material of said transformant, comprising a    protective antigen against protozoiasis development;-   [7] a method for producing a protective antigen against protozoiasis    development, comprising the step of isolating a protective antigen    against protozoiasis development from the transformed plant cell of    [2], the transformed plant of [3] or [4], or the propagation    material of [5];-   [8] a method for conferring immunity against a protozoan on an    animal, comprising the step of orally administering, to said animal,    the transformed plant cell of [2], the transformed plant of [3] or    [4], the propagation material of [5], the processed material or    extract of [6], or a protective antigen against protozoiasis    development obtained by the method of [7];-   [9] an oral anti-protozoiasis vaccine, comprising the transformed    plant cell of [2], the transformed plant of [3] or [4], the    propagation material of [5], the processed material or extract of    [6], or a protective antigen against protozoiasis development    obtained by the method of [7]; and-   [10] use of the transformed plant cell of [2], the transformed plant    of [3] or [4], the propagation material of [5], the processed    material or extract of [6], or a protective antigen against    protozoiasis development obtained by the method of [7], in producing    oral anti-protozoiasis vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of an ELISA on non-transgenic potatoes andtransgenic potatoes introduced with an R7 gene derived fromsecond-generation schizonts of Leucocytozoon caulleryi.

FIG. 2 is a photograph of electrophoresis showing the result of agenomic PCR performed on transgenic potatoes introduced with the R7 genederived from second-generation schizonts of Leucocytozoon caulleryi.

FIG. 3 shows the reinduction of blood antibodies in chickens that havebeen immunized by injection beforehand and have orally ingestedtransgenic potatoes introduced with the R7 gene derived fromsecond-generation schizonts of Leucocytozoon caulleryi.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides transformed plant cells introduced withgenes encoding protective antigens against protozoiasis development.

Protozoiasis is disease that develops when protozoans, which areunicellular eukaryotes, become parasitic on living bodies. In 1975, WHOnamed the six major tropical diseases malaria, trypanosomiasis,leishmaniasis, filariasis, schistosomiasis, and leprosy as diseases forwhich preventive measures are urgently needed. Three of these areprotozoan diseases and many protozoan diseases are rampant among bothhumans and animals.

Among them, infectious diseases caused by protozoans belonging toCoccidiasina Haemospororina include leucocytozoonosis caused byLeucocytozoon caulleryi infection and malaria (falciparum malaria,tertian malaria, quartan malaria, and ovale malaria) caused byPlasmodium protozoan infection. Infectious diseases caused byHaemoprotozoans belonging to Sarcomastigophora include leishmaniasis andtrypanosomiasis, and infectious diseases caused by Haemoprotozoans abelonging to Piroplasmia include babesiosis and theileriosis.

“Protective antigens against protozoiasis development” are antigens thatprotect against the development of such protozoiases. Examples includethe R7 antigen derived from second-generation schizonts of Leucocytozooncaulleryi (see amino acid sequence/SEQ ID NO: 1, and nucleotidesequence/SEQ ID NO: 2) against leucocytozoonosis, and AMA- 1 (apicalmembrane antigen 1) antigen (see Cheng, Q. and Saul, T., Mol. Biochem.Parasitol., 65:183-187 (1994)), CSP (circum sporozoite protein) antigen(see Haeseleer, F. et al., Mol. Biochem. Parasitol., 57:117-126 (1993)),LSA-1 (liver stage specific antigen-1) antigen (see Yang, C. et al.,Mol. Biochem. Parasitol., 71:291-294 (1995)), MSP-1 (merozoite surfaceprotein-1) antigen (see Ranford-Cartwright, L C. et al., Mol. Biochem.Parasitol., 46:185-187 (1991)), PfEMP1 (Plasmodium falciparum infectederythrocyte membrane protein 1) antigen (see Ward, C P. et al., Mol.Biochem. Parasitol., 102:167-177 (1999)), SERA (serine repeat antigen)antigen (see Li, W B. et al., Mol. Biochem. Parasitol., 33:13-25(1989)),and TRAP (thrombospondin related adhesion protein) antigen (seeTrottein, F. et al., Mol. Biochem. Parasitol., 74:129-141 (1995))against malaria, but the protective antigens of the present inventionagainst protozoiasis development are not limited thereto. Mutantprotective antigens with modified genetic sequences or amino acidsequences are also included in such protective antigens. Naturally orartificially produced mutant protective antigens are also included.

Plants derived from the transformed plant cells of the present inventionare not particularly limited so long as they are suitable for oraladministration, and examples include potato, tomato, beans such assoybean and adzuki (or “azuki”), grains such as rice, wheat and corn,fruits such as strawberry, and pasture grass.

The transformed plant cells of the present invention can be produced byintroducing a vector carrying a gene encoding the protective antigenagainst protozoiasis development into plant cells, and expressing thisgene. Vectors used for gene expression in plant cells are notparticularly limited so long as they include a promoter that can betranscribed in plant cells and a terminator sequence comprising apolyadenylation site necessary for stabilization of the transcriptionproduct, and examples include the “pBI121”, “pBI221”, and “pBI101”plasmids (all from Clontech). For example, as promoters that can betranscribed in plant cells, promoters for carrying out constitutive geneexpression in plant cells, or promoters that are inductively activatedby extrinsic stimuli may be used. Examples of promoters for constitutiveexpression include the 35S promoter of cauliflower mosaic virus (Odellet al. 1985 Nature 313:810), actin promoter of rice (Zhang et al. 1991Plant Cell 3:1155), and ubiquitin promoter of corn (Cornejo et al. 1993Plant Mol. Biol. 23:567). The protective antigen against protozoiasisdevelopment can be expressed inside plant cells by introducing intoplant cells, vectors carrying a gene encoding the protective antigenagainst protozoiasis development that is operably linked to suchpromoters. Herein, the phrase “operably linked” means that a promoterand a gene encoding a protective antigen against protozoiasisdevelopment are linked such that the protective antigen againstprotozoiasis development is expressed in the plant cells. The presentinvention also provides vectors for producing transformed plant cellscarrying such a protective antigen against protozoiasis development.

“Plant cells” that are transformed include plant cells having variousmorphologies such as suspension culture cells, protoplasts, leafsections, and calli.

Vectors can be introduced into plant cells using various methods knownto those skilled in the art, such as an Agrobacterium-mediated method,the polyethylene glycol method, electroporation method, and particle gunmethod.

A plant can be regenerated from a transformed plant cell byregeneration. Regeneration methods differ according to the type of plantcell, but a number of techniques have already been established andwidely used in the technical field of the present invention. Suchmethods include, for example, for potato, the method of Visser et al.(Theor. Appl. Genet 78:594 (1989)) or the tuber disc method for plantregeneration, for monocotyledonous cereals such as rice, the method ofHiei et al. (Hiei, Y., Komari, T., Kubo, T: Transformation of ricemediated by Agrobacterium tumefaciens. Plant Mol Biol 1997 35:1-2205-18), the method of Ishida et al. (Ishida, Y., Saito, H., Ohta, S.,Hiei Y, Komari, T., Kumashiro, T: High efficiency transformation ofmaize (Zea mays L.) mediated by Agrobacterium tumefaciens. NatBiotechnol 1996 June 14:6 745-50), electroporation method (Shimamoto,K., Terada, R., Izawa, T. et al.: Fertile transgenic rice plantsregenerated from transformed protoplasts. Nature 338, 274-276 (1989)),and for strawberry, the method of Asao et al. (Asao, H., Y Nishizawa, S.Arai, T. Sato, M. Hirai, K.

Yoshida, A. Shinmyo and T. Hibi.: Enhanced resistance against a fungalpathogen Sphaerotheca humuli in transgenic strawberry expressing a ricechitinase gene. Plant Biotechnology. 14 (3):145-149 (1997)). In thepresent invention, these methods can be preferably used.

Once a transformed plant that has a gene encoding a protective antigenagainst protozoiasis development introduced into its genome (chromosome)is obtained, progenies can be obtained by sexual or asexual reproductionfrom that plant. In addition, it is possible to obtain propagationmaterial (for example, seeds, fruits, grafts, tubers, tubercles,stubbles, calli, and protoplasts) from the plant, or from progenies orclones thereof, then use them as a source to mass-produce plants. Thepresent invention includes plants comprising the transformed plant cellsof the present invention, and progenies and clones thereof, as well aspropagation materials of the plants, and their progenies and clones.

Transformed plant cells, transformed plants, or propagation materialsthereof produced as described above may be used directly as vaccines fororal administration, but it is also possible to use their processedmaterials or extracts comprising the protective antigen againstprotozoiasis development. Herein, the term “processed materials” meanssubstances produced by processing transformed plant cells, transformedplants, or propagation materials thereof into a form suitable forvaccination. In the case of transgenic potato, an example is a materialproduced by freeze-drying potato leaves. The term “extract” means matterobtained by extracting components comprising a protective antigenagainst protozoiasis development from transformed plant cells,transformed plants, or propagation materials thereof, and includes thosethat are purified or crudely purified. Isolation (purification) ofprotective antigens against protozoiasis development from transformedplant cells, transformed plants, or propagation materials thereof can becarried out using conventional protein purification methods such as saltprecipitation, ultrafiltration, ion exchange chromatography and gelchromatography.

The present invention also provides methods for producing protectiveantigens against protozoiasis development, wherein the methods comprisesuch steps for isolating the protective antigens against protozoiasisdevelopment.

The present invention also provides methods for conferring immunityagainst protozoans on animals, wherein the methods comprise the step oforally administering the above-mentioned transformed plant cells,transformed plants, propagation materials, processed materials orextracts thereof, or protective antigens against protozoiasisdevelopment that were isolated by the above-mentioned method to theanimals.

In the present invention, the animals that are the object ofadministration of protective antigens against protozoiasis developmentare, for example, birds if the protozoiasis is leucocytozoonosis, andhumans, monkeys, and birds if it is malaria. Further examples includehumans and dogs for leishmaniasis, humans, cattle, and horses fortrypanosomiasis, dogs, cats, cattle, and horses for babesiosis, andcattle for theileriosis.

When orally administering a plant-body of the present invention carryinga protective antigen gene against protozoiasis development, theabove-mentioned transformed plant cells, transformed plants, propagationmaterials, processed material or extracts thereof, or the isolatedprotective antigens against protozoiasis development may be administeredalone or after mixing with other components. Examples of the othercomponents that may be used in combination include mucosal immuneadjuvants (for example, cholera toxin and cytokines), feeds of animalsthat are the object of administration, and additives (such as lactose)for improving palatability.

The dose differs depending on the animal that is the object ofadministration. For example, for chicken, mixing a plant carrying aprotective antigen gene against protozoiasis development with regularfeed, and feeding 2 to 5 g of this mixture per day, is thought to beconvenient. For other animals, an amount converted according to bodyweight, or an amount converted according to body surface area may beadministered, without limitation. Preferable doses can be determined bytaking into account the expression level of the preventive antigen,optimum antibody production, properties of the formula feed, and such.

Oral vaccines of the present invention can also be administered toanimals whose antibody titer has already been increased by vaccination(for example, vaccination by injection). This can further increase theantibody titer of such animals. For example, a significant increase ofantibody titer was confirmed in the present Examples when the vaccineswere orally administered to animals who had been inoculated withantigens expressed in E. coli and had high antibody titer level (animalswith an ELISA absorbance value of 0.4 or more, or preferably 0.7 ormore, in 3200-fold diluted serum).

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLES

Hereinbelow, the present invention will be more specifically describedwith reference to Examples, but it is not limited thereto.

Example 1 Insertion of An R7 Gene Encoding An Immunogenic ProteinDerived From Second-Generation Schizonts of Leucocytozoon Caulleryi IntoA Plant Expression Plasmid

The pBI-R7 expression vector of the present invention was obtained bydigesting the pTH-R7 plasmid vector comprising an R7 immunogenic proteingene derived from second-generation schizonts of Leucocytozoon caulleryi(JP-A H07-284392, and Granted/Registered Japanese Patent No. 3582663)using SmaI and SacI restriction enzymes, and cloning the obtained R7gene DNA fragments (SEQ ID NOs: 1 and 2) into the pBI121 plasmid vector(Clontech) carrying a cauliflower mosaic virus 35S promoter,β-glucuronidase gene, and nopaline synthase terminator, at the sitedigested using SmaI and SacI restriction enzymes.

Example 2 Introduction of the R7 Gene Derived From Second-GenerationSchizonts of Leucocytozoon Caulleryi Into Potato And Expression of theGene 1. Introduction of the R7 Gene Derived From Second-GenerationSchizonts of Leucocytozoon caulleryi Into Agrobacterium

The pBI-R7 plasmid for expressing the R7 gene derived fromsecond-generation schizonts of Leucocytozoon caulleryi, obtained asdescribed above in Example 1, was introduced into Agrobacteriumtumefaciens LBA 4404 (Clontech) by the direct introduction method usingfreeze-thaw treatment.

More specifically, Agrobacterium tumefaciens LBA 4404 was shake-culturedin 50 mL of LB liquid medium (1% Bactotryptone, 0.5% yeast extracts, 1%sodium chloride) at 28° C. until the absorbance at A600 reachedapproximately 1.0. After cooling on ice, the culture was centrifuged at3000 g at 4° C. (using Kubota RA-4), and bacterial cells were collectedand suspended in 1 mL of ice-cooled 20 mM calcium chloride solution. A0.1-mL aliquot of this suspension was placed in each Eppendorf tube.

One μg of the pBI-R7 recombinant plasmid was added and this was quicklyfrozen in liquid nitrogen. Next, the obtained frozen cells were thawedat 37° C. and then left to stand for 5 minutes. One mL of LB medium wasadded to these cells, and the mixture was shake-cultured at 28° C. for2-4 hours. After centrifugation at approximately 10,000 g for 1 minute(using Kubota KM-15200) to collect the bacterial cells, the cells weresuspended in 0.1 mL of LB medium, then spread onto a LB solid mediumcontaining rifampicin (100 μg/mL), kanamycin (25 μg/mL), andstreptomycin (300 μg/mL), and then cultured at 28° C. for 2-3 days toobtain transformed bacteria carrying pBI-R7.

The transformed Agrobacterium tumefaciens LBA4404 obtained above wasshake-cultured in LB liquid medium at 28° C. and then centrifuged at3000 g at 4° C. (using Kubota RA-6). The bacteria were then collected,suspended in MS medium [Physiol. Plant. 15:473 (1962)] and this was usedfor transformation of plants.

2. Introduction Into Potato By the Agrobacterium Method

The R7 gene derived from second-generation schizonts of Leucocytozooncaulleryi was introduced into potato by the tuber disc method using theAgrobacteria produced above.

More specifically, the skin of potato tubers were peeled, sterilized in1% sodium hypochlorite solution for 15 minutes, and washed six timeswith sterile distilled water. A sterilized cork borer was used to excisea 1-cm diameter cylindrical section from this tuber, and the section wassliced into 2-3-mm thick disks. These discs were soaked for 15 minutesin a MS liquid medium suspension of the Agrobacterium tumefaciensLBA4404 carrying pBI-R7 produced in section 1 above. They were thenincubated on MS medium [containing 3% sucrose, 0.1 μg/mL indole aceticacid, 0.1 μg/mL gibberellin, 0.1 μg/mL abscisic acid, and 2 μg/mL zeatinriboside (pH5.9)] at 28° C. for 3 days, and the discs were washed withMS liquid medium containing antibiotics (100 μg/mL of kanamycin and 500μg/mL of carbenicillin (both from Sigma)).

The washed discs were subcultured onto MS solid medium containing theabove-mentioned antibiotics (and also containing 3% sucrose) at 25° C.every two weeks (a 16-hour light period, and an 8-hour dark period). Onthe fourth to eighth week of culture, calli formed on the surface of thediscs, and further subcultures induced shoots.

These shoots were cut from the base, transplanted onto MS solid medium(containing 3% sucrose, 100 μg/mL of kanamycin, and 500 μg/mL ofcarbenicillin (pH5.9)) that does not contain hormones, and werecultured. Plants that took root 2 to 4 weeks later were transplanted toa 10-cm diameter pot containing potting compost, and were grown insidean artificial climate chamber.

3. Confirmation of Introduction of R7 Gene Derived FromSecond-Generation Schizonts of Leucocytozoon Caulleryi Into RegeneratedPlants And Verification of Gene Expression (1) Confirmation ofExpression of the R7 Gene Derived From Second-Generation Schizonts ofLeucocytozoon Caulleryi By ELISA

Regenerated potato leaves were ground in three times the wet volume ofPBS-T buffer (135 mM sodium chloride, 1.5 mM sodium dihydrogenphosphate, 2.7 mM disodium hydrogen phosphate, 0.05% (v/v) Tween20, pH7.2), and the supernatant obtained by centrifugation at 3000 g for 15minutes (using Kubota KS-5000) was used as crude sap.

Anti-Leucocytozoon caulleryi second-generation schizont monoclonalantibodies (Gotanda, T. et al., J. Vet. Med. Sci. 64 (3):281-283 (2002))diluted to a concentration of 2 μg/mL in 0.05 M sodium carbonate buffer[1.59 g of disodium carbonate and 2.93 g of sodium hydrogen carbonate inone liter (pH9.6)] were dispensed into each well of 96-well ELISA plates(IWAKI), and the plates were left to stand overnight at 4° C. forcoating.

The coated plates were washed with PBS-T buffer, then a blockingsolution prepared by adding bovine serum albumin to PBS-T buffer to afinal concentration of 3% (w/v) was dispensed into each well of theplates, and the plates were left to stand for one hour at 37° C. forblocking.

After the blocking treatment, the plates were washed with PBS-T buffer,and the crude sap of the leaves was added to the plates. The plates werethen left to stand for one hour at 37° C. to allow the reaction with theantibodies to take place. After washing these plates with PBS-T buffer,HRPO-labeled anti-Leucocytozoon caulleryi second-generation schizontmonoclonal antibodies [Ito, A. et al., J. Vet. Med. Sci. 64 (5):405-411(2002)] diluted 4000 times in an antibody dilution prepared by addingbovine serum albumin to PBS-T buffer to a final concentration of 0.3%(w/v) were dispensed to each well of the plates, and were allowed toreact for one hour at 37° C. After washing the plates with PBS-T buffer,a substrate solution (containing 14.6 g of disodium hydrogen phosphate,10.2 g of citric acid monohydrate, 1 g of o-phenylenediamine, and 1 mLof hydrogen peroxide solution in one liter) was added to the plates andallowed to react by leaving in the dark for 15 minutes at 37° C. Thereaction was stopped using a stop solution (5 N aqueous sulfuric acidsolution), and absorbance at A492 was measured on a microplate reader(using Corona MTP-120). As a result, the R7 protein derived fromsecond-generation schizonts of Leucocytozoon caulleryi was found to beexpressed in several regenerated potato plants (see FIG. 1).

(2) Confirmation of Introduction of the R7 Gene Derived FromSecond-Generation Schizonts of Leucocytozoon Caulleryi Into Potatoes

Leaves of several potato plants in which reactions were confirmed byELISA were ground into a powder in the presence of liquid nitrogen usinga mortar. RED Extract-N-Amp Plant PCR Kit (SIGMA) reagent was usedaccording to the manufacturer's protocol to extract the total DNA of theaforementioned potato leaves, and to perform genomic PCR using the R7fprimer (5′-GGAAATGTGTCCTTAACTTC-3′: SEQ ID NO: 3) and R7r primer(5′-CTTCTTCTTCATTACTTTTTC-3′: SEQ ID NO: 4) that specifically amplifythe R7 gene. When a portion of the reacted sample was subjected to 1.2%agarose gel electrophoresis, a band was observed at the same position asthat of the R7 gene, which confirmed that the R7 gene derived fromsecond-generation schizonts of Leucocytozoon caulleryi has beenintroduced into the chromosome in these potato plants (see theelectrophoresis photograph in FIG. 2: the arrow indicates the positionof the band for the R7 gene derived from second-generation schizonts ofLeucocytozoon caulleryi).

Example 3 Confirmation of the Antigenicity of Potatoes Introduced Withthe R7 Gene Derived From Second-Generation Schizonts of LeucocytozoonCaulleryi

The leaves of potatoes introduced with the R7 gene derived fromsecond-generation schizonts of Leucocytozoon caulleryi were freeze-dried(a 48-hour procedure using Kyowa Shinku Gijyutsu (Kyowa VacuumTechnology) RLE-204) until the dry weight became approximatelyone-twelfth that of the wet weight. The dried leaves were ground into apowder, then mixed with formula feed for poultry (Funabashi Farm), andused as an oral administration.

SPF chickens (Nisseiken Co. Ltd.) subjected to oral administration testswere three hens to which the leucocytozoonosis vaccine had been injected17 weeks earlier. The leucocytozoonosis vaccine is currently widely usedin the field, and uses an R7 protein derived from second-generationschizonts of Leucocytozoon caulleryi expressed in E. coli, as theantigenic component of the vaccine (JP-A H07-284392)). A total of 60 gof the oral administration (4 g of the total was the freeze-dried leavesof potato introduced with the R7 gene) was fed per day to each chicken,and the oral administration was continued for five consecutive days (thechicken were allowed to feed freely). After the period of feeding thefeed containing leaves of potato introduced with the R7 gene, 60 g offormula feed for poultry (Funabashi Farm) was fed per day to eachchicken. One chicken that was used as the control was given only 60 g ofthe ordinary formula feed for poultry per day from the beginning of theexperiment. The blood of all test chickens, including the controlchicken, was collected as needed from the wing vein starting from thetime when the oral administration (feeding) commenced, and the sera wereobtained by centrifugation. Using these sera, antibody tests werecarried out by the ELISA method described below.

Ultrasonicated and solubilized antigens from second-generation schizontsof Leucocytozoon caulleryi [Ito, A. et al., J. Vet. Med. Sci. 64(5):405-411 (2002)], which were diluted to a concentration of 0.1 μg/mLin 0.05 M sodium carbonate buffer [1.59 g of disodium carbonate and 2.93g of sodium hydrogen carbonate in one liter (pH9.6)], were dispensedinto each well of 96-well ELISA plates (IWAKI), and the plates were leftto stand overnight at 4° C. for coating.

The coated plates were washed with PBS-T buffer, then a blockingsolution prepared by adding bovine serum albumin to PBS-T buffer to afinal concentration of 3% (w/v) was dispensed into each well of theplates, and blocking performed by leaving the plates to stand for onehour at 37° C.

After the blocking treatment, the plates were washed with PBS-T buffer,and sera subjected to a two-fold stepwise dilution from 100-fold to51,200-fold using an antibody dilution prepared by adding bovine serumalbumin to PBS-T buffer to a final concentration of 0.3% (w/v) weredispensed into each well of the plates. The plates were then left tostand for one hour at 37° C. to allow the reaction with the antibodiesto take place. After washing these plates with a PBS-T buffer,HRPO-labeled anti-chicken IgG (available from ZYMED) diluted 12,000times with the antibody dilution was dispensed into each well of theplates, and was allowed to react for one hour at 37° C. After washingthe plates with PBS-T buffer, a substrate solution (containing 14.6 g ofdisodium hydrogen phosphate, 10.2 g of citric acid monohydrate, 1 g ofo-phenylenediamine, and 1 mL of aqueous hydrogen peroxide solution inone liter) was added to the plates and allowed to react by leaving theplates to stand in the dark for 15 minutes at 37° C. After stopping thereaction using a stop solution (5 N aqueous sulfuric acid solution), theabsorbance at A492 was measured on a microplate reader (Corona MTP-120).

The results of this experiment are shown in FIG. 3. As seen from FIG. 3,after oral administration of the R7 gene-introduced potato leaves,antibodies against second-generation schizonts of Leucocytozooncaulleryi were confirmed to be re-induced in all of the tested chickens.The increase was particularly significant in chickens having a higherantibody titer level at the start of the experiment.

INDUSTRIAL APPLICATION

The present invention provides (1) expression vectors for plant cells,which comprise a gene encoding a protective antigen against protozoiasisdevelopment, (2) transformed plant cells introduced with a gene encodinga protective antigen against protozoiasis development, (3) transformedplants comprising the transformed plant cells, and transformed plantsthat are the progenies or clones thereof, (4) propagation materials ofthe transformed plants, (5) processed material or extracts of thetransformants or propagation materials thereof, (6) methods forproducing protective antigens against protozoiasis development,comprising the step of isolating protective antigens againstprotozoiasis development from the transformed plant cells, thetransformed plants, or the propagation materials, and (7) methods forconferring immunity against a protozoan on animals, comprising the stepof orally administering an above-mentioned transformed plant cell,transformed plant, propagation material or processed material or extractthereof, or a protective antigen against protozoiasis developmentobtained by an above-mentioned method to these animals. The presentinvention has thus enabled the development of anti-protozoiasis vaccineswith superior cost performance that can be conveniently administered.

1. A vector comprising a gene encoding a protective antigen againstprotozoiasis development, wherein the gene is operably linked downstreamof a promoter that can be transcribed in a plant cell.
 2. A transformedplant cell introduced with comprising a gene encoding a protectiveantigen against protozoiasis development.
 3. A transformed plantcomprising the transformed plant cell of claim
 2. 4. A transformed plantwhich is a progeny or clone of the transformed plant of claim
 3. 5. Apropagation material of the transformed plant of claim 3 or
 4. 6. Aprocessed material or an extract of the transformant of claim 3 or 4, ora propagation material of said transformant, comprising a protectiveantigen against protozoiasis development.
 7. A method for producing aprotective antigen against protozoiasis development, comprisingisolating a protective antigen against protozoiasis development from thetransformed plant cell of claim 2, the transformed plant of claim 3 or4.
 8. A method for conferring immunity against a protozoan on an animal,comprising orally administering, to said animal, the transformed plantcell of claim 2, the transformed plant of claim 3 or
 4. 9. An oralanti-protozoiasis vaccine, comprising the transformed plant cell ofclaim 2, the transformed plant of claim 3 or
 4. 10. A method forproducing a protective antigen against protozoiasis development,comprising isolating a protective antigen against protozoiasisdevelopment from the propagation material of claim
 5. 11. A method ofconferring immunity against a protozoan on an animal, comprising orallyadministering, to said animal, the propagation material of claim
 5. 12.A method of conferring immunity against a protozoan on an animal,comprising orally administering, to said animal, the processed materialor extract of claim
 6. 13. A method of conferring immunity against aprotozoan on an animal comprising orally administering, to said animal,a protective antigen against protozoiasis development obtained by themethod of claim
 7. 14. An oral anti-protozoiasis vaccine, comprising thepropagation material of claim
 5. 15. An oral anti-protozoiasis vaccine,comprising the processed material or extract of claim
 6. 16. An oralanti-protozoiasis vaccine, comprising a protective antigen againstprotozoiasis development obtained by the method of claim 7.